Nucleic acid amplification is a crucial component of many techniques used in research, medicine, and industry. Such reactions are used in clinical and biological research, detection and monitoring of infectious diseases, detection of mutations, detection of cancer markers, environmental monitoring, genetic identification, detection of pathogens in biodefense applications, and the like, e.g. Schweitzer et al. Current Opinion in Biotechnology, 12:21-27 (2001); Koch, Nature Reviews Drug Discovery, 3:749-761 (2004). In particular, polymerase chain reactions (PCRs) have found applications in all of these areas, including applications for viral and bacterial detection, viral load monitoring, detection of rare and/or difficult-to-culture pathogens, rapid detection of bio-terror threats, detection of minimal residual disease in cancer patients, food pathogen testing, blood supply screening, and the like, e.g. Mackay, Clin. Microbiol. Infect., 10:190-212 (2004); Bernard et al. Clinical Chemistry, 48:1178-1185 (2002). In regard to PCR, key reasons for such widespread use are its speed and ease of use (typically performed within a few hours using standardized kits and relatively simple and low cost instruments), its sensitivity (often a few tens of copies of a target sequence in a sample can be detected), and its robustness (poor quality samples or preserved samples, such as forensic samples or fixed tissue samples are readily analyzed), Strachan and Read, Human Molecular Genetics 2 (John Wiley & Sons, New York, 1999).
Despite such advances in nucleic acid amplification techniques, there is still a need for further improvements, especially in applications that require rapid identification or quantification of critical markers, such as in infectious disease detection, minimum residual disease detection, bio-defense applications, intraoperative testing for surgical decisions, and the like, e.g. Raja et al. Clinical Chemistry, 48:1329-1337 (2002); Yoshioka et al. Surgery, 132:34-40 (2002); Qin et al. J. Clin. Microbiol., 41:4312-4317 (2003); Jaffe et al. J. Clin. Lab. Anal. 15:131-137 (2001).
In such applications, it is often important to amplify multiple sequences in a closed reaction vessel. These reaction conditions help to minimize false positives from contamination, allow the use of internal or external controls, allow measurement of sequences having widely varying abundances, and the like. To this end, techniques have been developed that allow for sequential amplification of multiple sequences in closed vessels by providing primer sets having widely differing annealing temperatures, e.g. Raja et al. (2002).
Despite the progress noted above, it would be highly useful for applications requiring rapid amplification of multiple sequences if additional methods were available for sequential multiplexing in amplification reactions, particularly if such methods could be combined with existing multiplexing approaches.